The present invention relates to drug resistance among infectious organisms or agents. More particularly, this invention relates to the use of antisense oligonucleotides to reverse such drug resistance, thereby resensitizing the infectious agents to therapeutic drugs.
A number of diseases are caused by infectious organisms which have become resistant to various chemotherapeutic drugs commonly used to treat such organisms. One such disease is malaria. Malaria is estimated to afflict more than 200 million people annually (WHO (1992) Bull. W.H.O. 70:801-804). While presently confined primarily to the tropics where it is endemic, malaria was formerly very wide spread, including the United States. However, in the late 1980""s small foci of periodic transmission of malaria in San Diego County were reported (Anonymous (1990) JAMA 263:1617), demonstrating that transmission capacity is still present in the United States, and that under the right circumstances, malaria could possibly become endemic once again.
Malaria is caused by infection with one or more species of Plasmodium. Three species (P. vivax, P. ovale, and P. malariae) produce relatively mild symptoms consisting of spiking periodic fever, anemia, and some jaundice. In contrast, infection with P. falciparum can lead to coma and death unless chemotherapy is initiated, and is responsible for about 800,000 deaths per year among African children under 5 years (WHO (1992) Bull. W.H.O. 70:801-804; and WHO (1992) WHO Weekly Epidemiological Record 22: p. 161-167).
Malaria infection is transmitted via the bite of an infected Anopheline mosquito during her blood meal. The sporozoite stage of the parasite is injected into the human host in mosquito saliva, and sporozoites then migrate to the liver where they invade hepatocytes, becoming intracellular parasites. Multiplication occurs within hepatocytes, which then rupture to release merozoite-stage parasites. These in turn invade circulating erythrocytes, beginning the asexual erythrocytic cycle of the parasite life cycle. It is the erythrocytic stages which are responsible for pathology to the human host. Within the erythrocyte, merozoites first develop into ring stage parasites, then trophozoites, then schizonts. Parasite DNA replication occurs during the trophozoite stage, giving rise to 16-20 merozoites at the end of schizogony. Mature schizonts cause the host erythrocyte to lyse, releasing merozoites which then reinvade erythrocytes to continue the cycle.
A small proportion of merozoites develop into male or female gametocytes. When drawn into a mosquito midgut during her blood meal, these erupt from the erythrocytes, fertilization occurs, and the zygotes penetrate the mosquito midgut wall to become oocysts. After asexual multiplication within oocysts, sporozoites are released, which migrate to the insect salivary glands to await the next mosquito blood meal. Injection of sporozoites in mosquito saliva during that next meal reinitiates the parasite life cycle.
Malaria has been treated with a variety of drugs, including anti-folate compounds such as pyrimethamine, trimethoprim, and proguanil (which inhibit the enzyme dihydrofolate reductase (Bzik et al. (1987) Proc. Natl. Acad. Sci. USA 84:8360-8364)), sulfonamides, (which inhibit dihydropteroate synthetase (Brooks et al. (1994) Eur. J. Biochem. 224:397-405), 4-aminoquinolines such as chloroquine (quinine analogs), sulfones, sulfanamides, and tetracyclines.
The antifolate drugs work by binding their target enzymes, thereby preventing normal enzyme function. While effective, resistance to these drugs can be mediated by selection for one or at most two point mutations which prevent binding of the drug to the active site (Brooks et al. (1994) Eur. J. Biochem. 224:397-405; Basco et al. (1995) Mol. Biochem. Parricidal. 69:135-138). Consequently, resistance to these drugs appeared fairly soon after these drugs were introduced (Peters, Chemotherapy and Drug Resistance in Malaria. (1987) London: Academic Press, pp. 15-20).
Until recently, chloroquine was by far the most commonly used antimalarial compound, owing to its low cost and lack of side effects compared with the antifolates (Peters, Chemotherapy and Drug Resistance in Malaria. (1987) London: Academic Press, pp. 5-14). However, after years of widespread chloroquine use, foci of resistant P. falciparum have been identified wherever malaria is endemic. Because of this widespread resistance, first antifolates, and now mefloquine have largely replaced chloroquine for treatment of P. falciparum and P. vivax as well (Peters, Chemotherapy and Drug Resistance in Malaria. (1987) London: Academic Press, pp. 659-670).
Mefloquine (a quinalone-methanol) has been shown to be effective against multi-drug resistant strains of P. falciparum (Harinasuta et al. (1983) Bull. WHO 61:299-305), and also been used for prophylactic use by travellers (Anonymous (1990) JAMA 263:2729-2737). While somewhat expensive and not without side effects, it remains the drug of choice for treating multi-drug resistant malaria (White (1988) Eur. J. Clin. Pharmacol. 34:1-14; and Anonymous (1990) JAMA 263:2729-2737). However, despite extensive measures to protect the efficacy of mefloquine, resistance has developed rapidly, and has even been found in areas where the drug has not been used clinically. In addition, wide spread cross resistance to other drugs has been demonstrated including structurally unrelated compounds such as halofantrine (Ringwald et al. (1990) Lancet 335:421-422; Gay et al. (1990) Lancet 336:1262; and Wilson et al. (1993) Mol. Biochem. Parricidal. 57:151-160), and artemesinin (Wilson et al. (1993) Mol. Biochem. Parricidal. 57:151-160), in addition to quinine (Brasseur et al. (1992) Am. J. Trop. Med. Hyg. 46:1-7; Brasseur et al. (1992) Am. J. Trop. Med. Hyg. 46:8-14; and Suebsaeng et al. (1986) Bull. WHO 64:759-765). The apparent cross-resistance to quinine is particularly significant because intravenous quinine remains the treatment of last recourse in cases of severe or cerebral malaria (Warrell et al. (1990) Trans. R. Soc. Trop. Med. Hyg. 84(suppl 2):1-65).
There is thus a need for improved chemotherapeutic drugs whose use inhibits or controls parasite infection without ultimately resulting in widespread resistance to such drugs and to those related thereto.
New chemotherapeutic agents have been developed which are capable of modulating cellular and foreign gene expression (see, Zamecnik et al. (1978) Proc. Natl. Acad. Sci. (USA) 75:280-284). These agents, called antisense oligonucleotides, bind to target single-stranded nucleic acid molecules according to the Watson-Crick rule or by other modes of hydrogen bonding, as well as base stacking, or to double stranded nucleic acids by the Hoogsteen rule of base pairing, or to and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of mRNA by RNase H, or by destroying the target via reactive groups attached directly to the antisense oligonucleotide.
Antisense oligonucleotides have been developed as antiparasitic agents, although none have been demonstrated to reverse drug resistant phenotype of a drug resistant parasite strain. PCT publication No. WO 93/13740 discloses the use of antisense oligonucleotides directed to nucleic acids encoding the dihydrofolate reductase-thymidylate synthase gene of P. falciparum to inhibit propagation of drug-resistant malarial parasites. Rapaport et al. (Proc. Natl. Acad. Sci. (USA) (1992) 89:8577-8580) teaches inhibition of the growth of chloroquine-resistant and chloroquine-sensitive P. falciparum in vitro using oligonucleotides directed to the dihydrofolate reductase-thymidylate synthase gene. PCT publication No. WO 94/12643 discloses antisense oligonucleotides directed to nucleic acids encoding a carbamoyl phosphate synthetase of P. falciparum. Tao et al. (Antisense Res. Dev. (1995) 5:123-129) teaches the uptake of antisense oligonucleotides by a schistosome parasite.
However, a need still remains for the development of oligonucleotides that are capable of inhibiting the replication of parasites and other infectious organisms. There is also a need for oligonucleotides which have the ability to reverse a drug resistant phenotype of a drug-resistant infectious organism, thereby resensitizing the organism to a therapeutic drug.
It is known that drug resistance among some infectious agents is mediated by enhanced expression of genes which actively subvert the activity of the relevant drugs, either by facilitating extracellular export of the drug (in the case of mdr-like genes), or by actively destroying the drug. It has been discovered that oligonucleotides specific for the pfmdr1 gene in the presence of mefloquine can reverse the drug-resistant phenotype of a mefloquine-resistant strain of parasite, thereby increasing parasite sensitivity to mefloquine. This discovery has important clinical consequences in facilitating continued use of mefloquine treatment in afflicted geographical areas where otherwise, mefloquine use is becoming increasingly restricted, due to decreased efficacy.
The discovery has been exploited to develop the present invention, which includes oligonucleotides, methods, and pharmaceutical formulations useful for reversing drug resistance and increasing parasite sensitivity to various chemotherapeutic drugs such as mefloquine, for inhibiting the expression of the pfmdr1 gene, and for treating diseases resulting from the infectious agent, such as malaria. The present invention represent a significant departure from previous applications of the antisense technology which have focused on the use of antisense oligonucleotides to directly inhibit growth and replication of infectious organisms.
In a first aspect, the invention provides a synthetic oligonucleotide having a nucleotide sequence complementary to a pfmdr1 nucleic acid. In one embodiment, the oligonucleotide of the invention is complementary to a region of pfmdr1 nucleic acid selected from the group consisting of a conserved region, an ATP binding site, a translational start site, and a Plasmodium falciparum-specific region.
As used herein, the term xe2x80x9csynthetic oligonucleotidexe2x80x9d includes chemically synthesized polymers of about five and up to about 50, preferably from about 19 to about 30 ribonucleotide and/or deoxyribonucleotide monomers connected together or linked by at least one, and preferably more than one, 5xe2x80x2 to 3xe2x80x2 internucleotide linkage.
For purposes of the invention, the term xe2x80x9coligonucleotide sequence that is complementary toxe2x80x9d a nucleic acid is intended to mean an oligonucleotide that binds to the nucleic acid sequence under physiological conditions, e.g., by Watson-Crick base pairing (interaction between oligonucleotide and single-stranded nucleic acid) or by Hoogsteen base pairing (interaction between oligonucleotide and double-stranded nucleic acid) or by any other means, including other forms of hydrogen bonding, base stacking, or in the case of an oligonucleotide binding to RNA, causing pseudoknot formation. Binding by Watson-Crick or Hoogsteen base pairing under physiological conditions is measured as a practical matter by observing interference with the function of the nucleic acid sequence.
The term xe2x80x9cregion,xe2x80x9d as used herein, refers to contiguous nucleotides making up a particular site, such as the translational start site or ATP binding cassette, as well as up to 20 nucleotides upstream and/or downstream from the site, or surrounding and including the site. The term xe2x80x9cconservedxe2x80x9d refers to nucleotide sequences within the gene of a species that are also found in other related or unrelated species or even genera. A xe2x80x9cPlasmodium falciparum-specific regionxe2x80x9d encompasses nucleotide sequences which are not found in other species.
In some embodiments the oligonucleotide of the invention has a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.
The oligonucleotides of the invention are modified in some embodiments. Preferred embodiments include at least one non-phosphodiester internucleotide linkage selected from the group consisting of phosphorothioates, phosphorodithioates, alkylphosphonates, alkylphosphonothioates, phosphoramidates, carbamates, acetamidates, carboxymethyl esters, carbonates, phosphate triesters, and combinations thereof in the case where more than one modified linkage is present. Particularly preferred embodiments have phosphorothioate internucleotide linkages. Some oligonucleotides of the invention include at least one ribonucleotide, at least one deoxyribonucleotide, or both. One preferred embodiment includes at least one 2xe2x80x2-O-alkylribonucleotide.
In another aspect, the invention provides a method of resensitizing an anti-malarial drug-resistant Plasmodium parasite to an anti-malarial drug, thereby reversing its drug-resistant phenotype. In this method, the parasite is cultured in the presence of a synthetic oligonucleotide complementary to a pfmdr-specific nucleic acid for a time sufficient to enable the oligonucleotide to hybridize to the nucleic acid. The parasite is then contacted and cultured with an anti-malarial drug in the presence of the oligonucleotide. In this way, the drug-resistant phenotype of the parasite can be reversed such that it can now be controlled by the drug. In preferred embodiments, the oligonucleotide used in the method of the invention are specific for a conserved region, an ATP binding site, a translational start site, or a Plasmodium falciparum-specific region of pfmdr1 nucleic acid, as described above. Anti-malarial drugs used in some embodiments are mefloquine, quinine, chloroquine, and/or derivatives thereof. In some preferred methods, mefloquine is used.
In another aspect, the invention provides a method of down-regulating the expression of pfmdr nucleic acid. In this method, the pfmdr nucleic acid is contacted with a synthetic oligonucleotide of the invention.
The invention also provides, in yet another aspect, a method of resensitizing a drug-resistant infectious organism to an anti-infectious organism drug, thereby reversing the drug-resistant phenotype of the organism. In this method, the infectious organism is cultured in the presence of a synthetic oligonucleotide complementary to a nucleic acid required for the drug-resistant phenotype for a time sufficient to enable the oligonucleotide to hybridize to the nucleic acid. In some embodiments, the oligonucleotide is complementary to pfmdr1 nucleic acid. The infectious organism is then contacted and further cultured with an anti-infectious organism drug in the presence of the oligonucleotide. In some embodiments, the infectious organism is a Plasmodium parasite, the oligonucleotide is complementary to pfmdr1 nucleic acid, and the drug is chloroquine, mefloquine, or quinine.
The pfmdr1-specific oligonucleotides of the invention are also useful for examining the function of the pfmdr1 gene in a control parasite and in a drug resistant parasite. Presently, gene function can only be examined by the arduous task of making a xe2x80x9cknock outxe2x80x9d animal. This task is difficult, time-consuming and cannot be accomplished for genes essential to animal development, since the xe2x80x9cknock outxe2x80x9d would produce a lethal phenotype. To date it has not been possible to increase or decrease gene copy number of pfmdr1 in malaria using gene transcription experiments. Thus, a direct demonstration of the function of the pfmdr1 gene in mefloquine resistance has not been shown. The present invention overcomes the shortcomings of this model.